µROX - µRNA Redox cycling

This project aims to develop a rapid, low-cost and ultrasensitive electrochemical lab-on-a-chip (LOC) platform comprising multiplexed microfluidics for the simultaneous detection of different microRNAs (RNAs) without any sample treatment. RNAs, small non-coding RNA molecules, play a potential role as diagnostic and prognostic biomarkers. Nowadays there are many different diagnostic platforms for the RNA profiling in centralized laboratories. However, due to their bulky and expensive equipment, and their need for a sample treatment along with a high sample volume they are not suitable for an application in point-of-care testing (POCT). To cover the demand for portable and fast point-of-care devices it is of great importance to realize a LOC platform allowing the measurement of multiple RNAs with fast sample-to-result times (e.g. without any sample preparation) along with a low sample consumption (e.g. blood from a finger prick). Microfluidic LOC platforms offer an acceleration of the specific and sensitive detection of various biomarkers. The proposed electrochemical RNA sensor is based on the combination of different amplification techniques. To increase the device sensitivity, interdigitated electrode arrays with gap sizes in the nanometer range are employed. In this regard, the technological limits will be investigated to achieve the possible highest amplification. In order to obtain enzyme-mediated signal amplification, the stop-flow technique is utilized. A further signal amplification is achieved by DNA or RNA strains and/or by an appropriate surface modification to increase the number of binding sites. The combination of these amplification approaches represents a drastic increase in sensitivity compared to the conventional electrochemical methods and allows a fast and reliable measurement of RNAs without prior amplification by means of polymerase chain reaction (PCR) techniques. One of the specific applications for RNAs, where only a low sample volume is available, is the aggressive brain tumor in young children (earliest incidence 18 month), known as Medulloblastoma (MB). Beside the histopathologic classification of MB, it can be divided into four molecular subtypes. An early classification of these subtypes or detection of relapse will increase the survival chances of patients. This proposal is dedicated to implement a new detection method for cancer research using a unique interplay of different research fields (Micro-Nanotechnology, microfluidics and biomedicine). A successful demonstration of the multiplexed RNA profiling system will have a great impact on clinical diagnostics, especially in POCT, and will be a low-cost and user-friendly alternative to standard PCR systems and other diagnostics systems. Furthermore, it will be possible to measure RNAs not only in samples like blood plasma, saliva and urine, but also directly in undiluted serum or even whole blood.

The aim of this project was to develop a fast and ultra-sensitive electrochemical lab-on-a-chip (LOC) platform that simultaneously detect different microRNAs (RNAs) without sample preparation. The project consisted of three main groups: The production of the microchip with nanogaps, the finding of potential biomarkers (RNAs) for tumor classification and the development of a highly sensitive and selective detection method. The microchip, which has finger-like structures in the nanometer range, is manufactured using the manufacturing technologies of the semiconductor industry. These finger-like structures serve as electrodes for the electrochemical measurement. A wide variety of materials (Pt, Au, carbon) were used as electrodes and the electrochemical behavior was studied. A special electrochemical method, in which certain substances are oxidized and reduced again, was used. These substances behave differently depending on the electrode material. The advantage is that a signal amplification can be achieved in comparison to the only oxidized or only reduced sensors. The smaller the distance between the electrodes, the higher the signal amplification. Our tests have shown that this amplification depends very much on the electrode material used and on the substance and that the largest constant signal amplification window can be achieved using gold. A signal amplification of 161-fold was determined for the gold nanogap chip with FcMeOH as a redox couple at an electrode distance of 100 nm. Carbon as an electrode material, on the other hand, has a non-constant gain window, which limits the area of application. Different combinations of redox substances and electrode materials were investigated in order to obtain maximum signal amplification. The focus was not only on signal amplification, but also on stability and the possible measuring range. In addition, potential biomarkers, a total of 50 highly aggressive brain tumors (medulloblastomas and ETMR) from small children, were examined and more than 870 tumor-associated RNAs per sample were analyzed using a "high throughput" method. A high regulated RNA cluster was identified for both tumor identities and subsequently also found in the serum samples of the patients with an increased value. In addition, the detected values could also be correlated with the course of the tumor. The more aggressive and resistant a tumor was, the higher the corresponding RNAs were detectable in the patient's serum. In order to detect the RNAs, a system is required that handles the liquids and can specifically detect the RNAs with a high sensitivity. This fluidic system (LOC) must also contain the method that detects the specific RNA very sensitively and very selectively. In this project, a new type of electrochemical system based on CRISPR/Cas13a was developed. This makes it possible to detect the desired RNAs very selectively.